Silencing CAMK2G and CAMK2D in HRMECs

HRMECs were transfected with Dharmacon ON-TARGETplus SMARTpool siRNAs specifically targeting human CAMK2G (catalog L-004536-00) or CAMK2D (catalog L-004042-00) isoforms for 24 hours using DharmaFECT reagent (Thermo Fisher Scientific). Dharmacon ON-TARGETplus siCONTROL Nontargeting siRNA (catalog D-001810-10) was used as control siRNA. Quantitative PCR (qPCR) was performed to determine knockdown efficiency as previously described (66 (link)). Primers (Integrated DNA Technologies) were designed to amplify human CAMK2G (forward primer 5′-TCCTGTATATCCTCCTGGT-3′, reverse primer 5′-CATCTGGTTGATCAAGTTC-3′) and human CAMK2D (forward primer 5′-GGATCTGTCAACGTTCTACT-3′, reverse primer 5′-TGTGGATTACAGTAGTTTGG-3′), which were quantified relative to GAPDH (human, forward primer 5′-GAGTCAACGGATTTGGTCGT-3′, reverse primer 5′-GACAAGCTTCCCGTTCTCAG-3′). Cells were transfected with 25 nM siRNA, a concentration that was found to consistently produce ≥80% transcript knockdown for each CAMKII isoform and where knockdown of CAMK2G had no effect on CAMK2D mRNA expression and vice versa (Supplemental Figure 6).